WO2013120620A2

WO2013120620A2 - Combination of n-cocoyl alanine and of an extract of malus domestica dedifferentiated cells

Info

Publication number: WO2013120620A2
Application number: PCT/EP2013/000449
Authority: WO
Inventors: Anne-Cécile HARIVEL
Original assignee: Svr Recherche
Priority date: 2012-02-16
Filing date: 2013-02-15
Publication date: 2013-08-22
Also published as: FR2986967A1; WO2013120620A3

Abstract

The present invention relates to the use of a combination of N-cocoyl alanine and a suspension of Malus domestica dedifferentiated plant cells as a cosmetic active agent which regulates the multiplication and/or differentiation capacity of epidermal stem cells, to a method of non-therapeutic treatment of the human body using such a regulating cosmetic active agent and to a cosmetic composition comprising such an active agent.

Description

COMBINATION OF N-COCOYL ALANINE AND EXTRACT FROM DEDIFFERENCED CELLS

FROM MALUS DOMESTICA

The present patent application claims the priority of the French patent application FR 1,200,440 filed on February 16, 2012, which is incorporated herein by reference. The present invention relates to the field of cosmetic and pharmaceutical active ingredients as well as to topical compositions containing them.

Stem cells (SC) are undifferentiated cells with the property of regenerating and differentiating into cells of any cell type. Because of its properties, these cells have since their discovery a central object of research on multiple applications ranging from cell therapy to plastic surgery.

SCs can be divided into two groups corresponding to embryonic stem cells and adult stem cells.

Embryonic stem cells play a central role during the first phase of development of the organism where they are at the base of the establishment of each cell type. In a way, they have the capacity to form a complete organism from a single cell type which is called pluripotent cells. Unfortunately, the existence of these cells is limited to the embryonic stages and they are therefore absent in later stages of life.

The second type of stem cells are adult stem cells, which can be identified in many adult tissues or organs such as the spinal cord, pancreas, central nervous system, dental pulp, heart or dermis. In comparison with embryonic stem cells, adult stem cells have more limited potential in terms of differentiation. Some of them can not be differentiated into a single cell type, most often the one surrounding them. These cells are then called unipotent stem cells. Other adult stem cells, failing to differentiate into cells of any cell type have at least the ability to differentiate into several distinct cell types. These adult stem cells are described as multipotent.

These two types of adult stem cells are promising for medical uses because they are more accessible and obtaining them is less ethically problematic than those of embryonic stem cells.

The skin is a multi-laminar system composed of two major tissues: the dermis and the epidermis in contact with the outside, between which is intercalated a junction zone called dermal-epidermal junction (JDE), with:

- The dermis is a dense and fibroelastic connective tissue made up of the extracellular matrix (ECM), whose production and eventual remodeling are essentially provided by the fibroblasts.

- The dermo-epidermal junction (JDE) is the junction zone between the basal keratinocytes of the epidemic and the dermis. It is characterized by the presence of a basement membrane, in which the extracellular matrix forms a thin and solid lattice. It consists of five major components corresponding to type IV collagen, laminins, heparan sulfate, nidogen and fibronectin.

- The epidermis is essentially composed of keratinocytes that proliferate in the basal layer and then gradually differentiate to give the different layers of the epidermis by migrating from the depth to the surface, where they desquamate. Adult skin, like other organs of the body, contains stem cells that regulate its renewal, surface appearance, and even changes over time. Among these stem cells, there are pluripotent follicular stem cells and epidermal stem cells, which are characterized by a strong ability to self-renew on the one hand and on the other hand, to generate highly differentiated daughter cells. In the epidermis, the stem cells are located within the basal layer and they represent 1 to 10% of the basal cells.

These stem cells are essential components for the processes of skin healing and regeneration of hair and skin. However, the capacities of these stem cells, and in particular that of the epidermal stem cells are affected by many parameters among which aging is accompanied by a decrease in the number and the capacity of division and differentiation of these cells.

In order to protect skin stem cells, various "anti-aging" assets could be developed including the SURVICODE ^® corresponding to an N-cocoyl alanine which maintains the survival of stem cells in the epidermis.

The inventors have now demonstrated that a combination of N-cocoyl alanine and a suspension of dedifferentiated plant cells of Malus domestica has a synergistic action on the capacity for multiplication and / or differentiation of epidermal stem cells.

Accordingly, a first object of the invention relates to the use of a combination of N-cocoyl alanine and a suspension of dedifferentiated plant cells of Malus domestica as a cosmetic active regulator of the multiplication capacity and / or differentiation of epidermal stem cells. The N-cocoyl alanine used in the uses as defined above may be in free acid form or in partially or totally salified form. When it is in salified form, it is in particular alkali salts such as sodium, potassium or lithium salts, alkaline earth salts such as calcium, magnesium or strontium salts; ammonium salt or salt of an aminoalcohol such as (2-hydroxyethyl) ammonium salt. It may also be metal salts such as divalent salts of zinc or manganese, trivalent salts of iron, lanthanum, cerium or aluminum. In general, the degree of salification of said N-acyl amino acid will depend in addition on its PK _A and the salt concentration of the composition in which it is incorporated.

N-cocoyl alanine is preferably a mixture of N-acyl alanine obtained by acylation of alanine with activated derivatives of fatty acids derived from coconut oil.

A dedifferentiated plant cell suspension of Malus domestica can be obtained simply by those skilled in the art. Briefly, a stable differentiated cell line can be obtained simply by performing the steps of selecting a suitable piece of Malus domestica tissue, sterilizing the surface of that piece of tissue, laying a tissue explant on a solid medium adapted for the induction of calli, callus harvesting, subculture thereof in a suitable medium so as to obtain fully dedifferentiated cells, addition of dedifferentiated cells in a reactor comprising a suitable liquid culture medium and culture of these with agitation to maintain the homogeneity of the crop.

Methods and technologies for plants are well known to those skilled in the art. By way of example, working protocols relating to plant cultures can be found in "Plant Cell Culture" (A Practical Approach, PA Editor Dixon, 1994, Oxford University Press), protocols and adapted culture media. for initiation of apple plant cell cultures in NITSCH et al. (Physiological bases of apple and pear meat production in vitro, Bull Soc., Fr., vol.117, p: 479 to 492, 1970) or in PECH ef. al. (In Vitro Growth of Apple Cell Tissues and Suspensions, Bull Soc.Brok Fr., vol.122, p: 183 to 194, 1975). The production of biomass is also carried out according to methodologies well known to those skilled in the art. Thus, the suspension culture obtained is used as an inoculum for the next culture from an Erlenmeyer flask to culture reactors of 50 to 100 liters. In this context, from 5 to 10% of the volume of the suspension is used as inoculum for the next culture. Finally, the dedifferentiated plant cell suspension of Malus domestica undergoes lysis, preferably mechanical lysis simply by mechanical lysis (eg, sonication or rapid agitation) or osmotic shock. In addition to extraction, care should be taken to add suitable preservatives (eg phenoxyethanol, benzoic acid, propionic acid, alcohol, silver chloride, ascorbic acid or tocopherol). Advantageously, the lysis will be carried out by homogenization under high pressure allowing the destruction of cell membranes. The lysate obtained can be put into the form of a dry extract by means of freeze-drying.

More simply, the skilled person can obtain such a cell suspension by following all or part of the process steps as described in patent application US 2008/0299092.

Advantageously, said suspension of dedifferentiated plant cells of Malus domestica will take the form of a dry extract (powder).

Preferably, the term Malus domestica, the cultivar Uttwiler Spaetlauber.

The subject of the invention is also a method for non-therapeutic treatment of the human body intended to maintain the capacity for multiplication and / or differentiation of epidermal stem cells in human skin, characterized in that a composition containing a cosmetically acceptable medium and an effective amount of a combination of N-cocoyl alanine and a suspension of Dedifferentiated plants of Malus domestica as defined above.

In the uses and in the process as defined above, the N-cocoyl alanine and the dedifferentiated plant cell suspension of Malus domestica is generally used in a composition intended for topical application to the skin or even the mucous membranes. Said composition intended for a topical application comprises between 0.1% and

10% by weight, more particularly between 0.5% to 5% by weight, and especially between 0.5% and 1.5% by weight of N-cocoyl alanine.

Said composition intended for topical application comprises between 0.05% and 5% by weight, more particularly between 0.1% to 1% by weight, and very particularly between 0.2% and 0.5% by weight of a dry extract. of a dedifferentiated plant cell suspension of Malus domestica.

Said treatment or said uses are implemented by applying said composition intended for topical application on the surface of the skin or mucosa to be treated. The compositions used in said process or in said uses are generally in the form of dilute aqueous or hydroalcoholic solutions, in the form of single or multiple emulsions, such as water-in-oil (W / O) emulsions, oil in water (O / W) or water in oil in water (W / O / W), in which the oil is vegetable, synthetic or mineral in nature, or in powder form. The compositions used in said treatments are administered to the subject in the conventional forms used in cosmetics and pharmacy; it is more particularly the topical, oral or parenteral administrations.

In general, the combination according to the invention may be associated with many types of adjuvants or active ingredients used in cosmetic formulations, whether it be fatty substances, organic solvents, thickeners or gelling agents. , softeners, antioxidants, opacifiers, stabilizers, foaming and / or detergent surfactants, emollients, superfatting agents, fragrances, ionic or non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, essential oils, dyestuffs, pigments, hydrophilic or lipophilic active agents, humectants, for example glycerine or glycols, preservatives, dyes, cosmetic active ingredients, mineral sunscreens and / or organic, mineral fillers such as iron oxides, titanium oxides and talc, synthetic fillers such as nylons and poly (methyl methacrylate) crosslinked or not, silicone elastomers, seric ites or extracts of plants or lipid vesicles, self-tanning agents such as DHA and / or erythrulose, oxidants such as hydrogen peroxide, or any other ingredient usually used in cosmetics.

Examples of oils that may be mentioned are paraffins, isoparaffins, mineral white oils, vegetable oils (from flowers, fruits, vegetables, trees, cereals, oilseeds, etc.). animal oils, synthetic oils, silicone oils and fluorinated oils; and more particularly: oils of vegetable origin, such as sweet almond oil, coconut oil, castor oil, jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil, wheat germ oil, corn germ oil, soybean oil, cottonseed oil, alfalfa oil, lemon poppy oil, pumpkin oil, evening primrose oil, millet oil, barley oil, rye oil, carthame oil, passionflower oil, hazelnut oil, palm oil, shea butter, apricot kernel oil, calophyllum, sysymbrium oil, avocado oil, calendula oil, oils derived from flowers or vegetables; ethoxylated vegetable oils; oils of animal origin, such as squalene, squalane; mineral oils, such as paraffin oil, liquid petrolatum and isoparaffins; synthetic oils, especially fatty acid esters such as butyl myristate, propyl myristate, cetyl myristate, isopropyl palmitate, butyl stearate, hexadecyl stearate, isopropyl stearate octyl stearate, isocetyl stearate, dodecyl oleate, hexyl laurate, propylene glycol dicaprylate; esters derived from lanolic acid, such as isopropyl lanolate, isocetyl lanolate, monoglycerides, diglycerides and triglycerides of fatty acids such as glycerol triheptanoate, alkylbenzoates, polyalphaolefms, polyolefins such as polyisobutene, synthetic isoalkanes such as isohexadecane, isododecane, perfluorinated oils and silicone oils. Among these, mention may be made more particularly of dimethylpolysiloxanes, methylphenylpolysiloxanes, amine-modified silicones, silicones modified with fatty acids, silicones modified with alcohols, silicones modified with alcohols and fatty acids, and modified silicones. by polyether groups, modified epoxy silicones, silicones modified with fluorinated groups, cyclic silicones and silicones modified with alkyl groups.

Other fatty substances that may be mentioned include linear or branched, saturated or unsaturated fatty alcohols, linear and / or branched, saturated and / or unsaturated fatty alcohol mixtures, or linear or branched, saturated or unsaturated fatty acids. unsaturated, mixtures of linear or branched, saturated or unsaturated fatty acids.

Among the thickening and / or emulsifying polymers that may be used, there are, for example, homopolymers or copolymers of acrylic acid or derivatives of acrylic acid, homopolymers or copolymers of methacrylic acid or derivatives of methacrylic acid. , homopolymers or copolymers of acrylamide, homopolymers or copolymers of acrylamide derivatives, homopolymers or copolymers of the acid acrylamidomethyl propanesulfonic acid, homopolymers or copolymers of vinyl monomers, homopolymers or copolymers of trimethylaminoethylacrylate chloride, hydrocolloids of vegetable or bio synthetic origin such as, for example, xanthan gum, karaya gum, carrageenates, alginates; silicates; cellulose and its derivatives; starch and its hydrophilic derivatives; polyurethanes.

Among the polyelectrolyte type polymers that can be used in the production of a gelled aqueous phase that can be used in the preparation of W / O, O / W, E / H / E or O / W emulsions, or an aqueous gel comprising SEPIBIO ™ POTENTILLA 217, there are, for example, copolymers of acrylic acid and 2-methyl-[(1-oxo-2-propenyl) amino] -acrylic acid. propane sulfonic acid (AMPS), copolymers of acrylamide and 2-methyl-[(1-oxo-2-propenyl) amino] 1-propane sulfonic acid, copolymers of 2-methyl [(1-Oxo-2-propenyl) amino] 1-propanesulfonic acid and (2-hydroxyethyl) acrylate, the homopolymer of 2-methyl-[(1-oxo-2-propenyl) amino] 1-propane sulfonic acid, homopolymer of acrylic acid, copolymers of acryloyl ethyl trimethyl ammonium chloride and acrylamide, copolymers of AMPS and vinylpyrrolidone, copolymers of AMPS and N ₅ N dimethyl acrylamide, the terpolymers of AMPS, of acrylic and N ₅ N dimethyl acrylamide, copolymers of acrylic acid and alkyl acrylates whose carbon chain comprises between ten and thirty carbon atoms, copolymers of AMPS and alkyl acrylates whose carbon chain comprises between ten and thirty carbon atoms.

Among the waxes that can be used in the compositions according to the invention, mention may be made, for example, of beeswax, carnauba wax, candelilla wax, ouricoury wax, japanese wax and cork fiber wax. or sugar cane, paraffin waxes, lignite waxes, microcrystalline waxes, lanolin wax, ozokerite, polyethylene wax, hydrogenated oils, silicone waxes, vegetable waxes, fatty alcohols and the solid fatty acids at room temperature, solid glycerides at room temperature.

Among the emulsifiers that may be used in the compositions according to the invention, mention may be made of: the fatty esters of alkylpolyglycosides which may be alkoxylated;

alkoxylated fatty esters;

carbamates of polyalkylene glycols with fatty chains;

fatty acids, ethoxylated fatty acids, fatty acid and sorbitol esters, ethoxylated fatty acid esters, polysorbates, polyglycerol esters, ethoxylated fatty alcohols, sucrose esters, alkylpolyglycosides, sulphated and phosphated fatty alcohols or mixtures of alkylpolyglycosides and fatty alcohols;

the combinations of emulsifying surfactants chosen from alkylpolyglycosides

associations of alkylpolyglycosides and fatty alcohols, esters of polyglycerols or polyglycols or polyols.

Among the foaming and / or detergent surfactants that can be used in the compositions according to the invention, mention may be made of: the topically acceptable anionic, cationic, amphoteric or nonionic surfactants usually used in this field of activity.

Among the anionic surfactants that may be used in the compositions according to the invention, mention may be made especially of alkali metal salts, alkaline earth metal salts, ammonium salts, amine salts, and aminoalcohol salts of the following compounds alkyl ether sulphates, alkyl sulphates, alkyl amido ether sulphates, alkyl aryl polyether sulphates, sulphate monoglycerides, alpha-olefin sulphonates, paraffinsulfonates, alkylphosphates, alkyletherphosphates, alkylsulfonates, alkylamidesulfonates, alkylarylsulfonates, alkylcarboxylates, alkylsulfosuccinates, alkylethersulfosuccinates, alkylamidesulfosuccinates, alkylsulfoacetates, alkylsarcosinates, acylisethionates, N-acyltaurates, acyllactylates.

Among the amphoteric surfactants that may be used in the compositions according to the invention, mention may be made of alkyl betaines, alkylamidobetaines, sultaines, alkylamidoalkylsulfobetaines, imidazoline derivatives, phosphobetaines, amphopolyacetates and amphopropionates. In order to further potentiate the activity obtained by the combination according to the invention, it may be combined with other active agents, especially those known for their antioxidant, antiradical, anti-aging, firming, restructuring, stimulating, energizing, oxygenating action. , anti wrinkle, relaxing, moisturizing, emollient, anti-dandruff, antimicrobial, seborrégulatrice, purifying, soothing, relaxing, relaxing, anti-stress, slimming, draining, lightening, depigmenting or propigmenting, neuromodulator, immunomodulator, tensor, filler, exfoliator, stimulator cellular renewal, lifting, plumping, improving radiance, etc.

The combination according to the invention may also be associated with compounds having a sensory property, such as a cooling effect (example mint extracts, alcohol ...) or heating effects to optimize skin comfort. .

The following experimental study illustrates the invention without limiting it. It shows that a combination of N-cocoyl alaninate and an extract of a plant stem cell suspension derived from Malus domestica has a synergistic effect on maintaining the capacity for growth and / or differentiation of keratinocyte stem cells.

1) Maintenance of the growth capacity of keratinocyte stem cells

Demonstration of the synergistic action of a combination of N-cocoyl alaninate and an extract of plant stem cells derived from Malus domestica on the maintenance of the ability of keratinocytes to form colonies in a differentiated epidermis model.

This differentiated epidermis model is achieved simply by starting from

keratinocytes isolated from the foreskin as described in particular in Yamada (Current Protocol in Cell Biology, "Preparation of Human Epidermal Kerotinocyte Cultures", 2004). Briefly, keratinocytes are isolated from human foreskin and are then inoculated for 5 minutes on a collagen-coated culture medium in the presence of a culture medium for the keratinocytes. The culture is then subjected to various washes so as to eliminate loose cells.

The culture of keratinocytes is then cultured for a period of one week in the presence or absence of N-cocoyl alaninate and an extract of plant stem cells derived from Malus domestica prepared as described in US patent application 2008 / 0299092.

The cultures are then trypsinized and re-seeded at low density (2000 cells per well, n = 5) in a 96-well plate. The cells are then cultured again in the presence or absence of N-cocoyl alaninate and a liposomal preparation of plant stem cells derived from Malus domestica until it reaches a maximum of 30-50% confluence.

Finally, the morphology and the number of colonies under a microscope (INCELL ANALYZER 1000) are determined. In addition, and to determine the function

mitochondrial and viability, the cells are incubated in the presence of bromide of 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium (MTT, SIGMA) for at least 3 hours at 37 ° C. The dark bluish crystals formed are observed by microscopy (INCELL ANALYZER 1000) and the crystals are lysed in the presence of a solution of SDS acid (0.01N HCl in 10% SDS) and the absorbance is measured at 570 nm.

2) Slow aging of keratinocyte stem cells

Demonstration of the synergistic action of a combination of N-cocoyl alaninate and an extract of plant stem cells derived from Malus domestica on slowing the aging of keratinocyte stem cells in a differentiated epidermis model.

A keratinocyte culture is carried out as above and incubated in the presence or absence of N-cocoyl alaninate and an extract of plant stem cells derived from Malus domestica prepared as described in US patent application 2008/0299092.

The different cultures are maintained for 6 passages (approximately 6 weeks) under the same culture conditions.

At this term, the cells are trypsinized and seeded in nacelle (n = 2). The cells are maintained for about 12 days so as to allow their differentiation into reconstructed epidermis.

Finally, the differentiation of cultures is analyzed using two markers: loricrine as a late marker and claudin-1 as a marker of intercellular junction quality.

Claims

1. Use of a combination of N-cocoyl alanine and a dedifferentiated plant cell suspension of Malus domestica as a cosmetic active regulator of the ability to multiply and / or differentiate epidermal stem cells.

2. Use according to claim 1, wherein the N-cocoyl alanine corresponds to the mixture of N-acyl alanine obtained by acylation of alanine with activated derivatives of fatty acids from coconut oil.

3. Use according to any one of claims 1 or 2, characterized in that the dedifferentiated cells of Malus domestica are from the cultivar Uttwiler Spaetlauber.

4. Use according to any one of claims 1 to 3, characterized in that the suspension of dedifferentiated plant cells of Malus domestica are in the form of a dry extract.

5. Use according to claim 4, characterized in that said combination comprises between 0.1% to 1% by weight, preferably between 0.2% and 0.5% by weight, of a dry extract of a cell suspension. of dedifferentiated plants of Malus domestica.

6. Use according to any one of claims 1 to 5, characterized in that said combination comprises between 0.1% and 10% by weight, preferably between 0.5% to 5% by weight of N-cocoyl alanine.

7. A method of non-therapeutic treatment of the human body intended to maintain the capacity for multiplication and / or differentiation of epidermal stem cells, characterized in that a composition containing a cosmetically acceptable medium and an effective amount is applied to the epidermis. of a combination of N-cocoyl alanine and a suspension of plant cells

Dedifferentiated derivatives of Malus domestica as defined in any one of Claims 1 to 6.

A cosmetic composition for maintaining the ability to multiply and / or differentiate epidermal stem cells comprising a combination of N-cocoyl alanine and a dedifferentiated plant cell suspension of Malus domestica as defined in any one of Claims 1 to 3.